Water channel proteins in bile formation and flow in health and disease: When immiscible becomes miscible

Molecular Aspects of Medicine 33 (2012) 651–664

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Molecular Aspects of Medicine journal homepage: www.elsevier.com/locate/mam

Review

Water channel proteins in bile formation and flow in health and disease: When immiscible becomes miscible Piero Portincasa a,⇑, Giuseppe Calamita b,⇑ a b

University of Bari Medical School, Clinica Medica ‘‘A. Murri’’, Department of Biomedical Sciences and Human Oncology, Policlinico Hospital, 70124 Bari, Italy Department of Biosciences, Biotechnologies and Pharmacological Sciences, University of Bari, Via Amendola 165/A, 70126 Bari, Italy

a r t i c l e

i n f o

Article history: Available online 7 April 2012 Keywords: Aquaporins Bile flow Bile acids Cholesterol Membrane Liver Water absorption Water secretion

a b s t r a c t An essential function of the liver is the formation and secretion of bile, a complex aqueous solution of organic and inorganic compounds essential as route for the elimination of body cholesterol as unesterified cholesterol or as bile acids. In bile, a considerable amount of otherwise insoluble cholesterol is solubilized by carriers including two other classes of lipids, namely phospholipid and bile acids. Formation of bile and generation of bile flow are driven by the active secretion of bile acids, lipids and electrolytes into the canalicular and bile duct lumens followed by the parallel movement of water. Thus, water has to cross rapidly into and out of the cell interior driven by osmotic forces. Bile as a fluid, results from complicated interplay of hepatocyte and cholangiocyte uptake and secretion, concentration, by involving a number of transporters of lipids, anions, cations, and water. The discovery of the aquaporin water channels, has clarified the mechanisms by which water, the major component of bile (more than 95%), moves across the hepatobiliary epithelia. This review is focusing on novel acquisitions in liver membrane lipidic and water transport and functional participation of aquaporin water channels in multiple aspects of hepatobiliary fluid balance. Involvement of aquaporins in a series of clinically relevant hepatobiliary disorders are also discussed. Ó 2012 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5.

6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biological relevance of membrane water transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aquaporins, highways for cells to recycle water with the outside world. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hepatic secretion of biliary lipids and bile formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Distribution, regulation and physiology of biliary aquaporins in mammals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Hepatocyte express multiple aquaporins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.1. AQP8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.2. AQP9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.3. AQP11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.4. AQP3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Aquaporins of the intrahepatic bile ducts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Gallbladder aquaporins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. AQPs of hepatobiliary endothelial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathophysiology of biliary aquaporins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

⇑ Corresponding authors. Tel.: +39 0805478234; fax: +39 0805478232 (P. Portincasa), tel.: +39 0805442928; fax: +39 0805443388 (G. Calamita). E-mail addresses: [email protected] (P. Portincasa), [email protected] (G. Calamita). 0098-2997/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mam.2012.03.010

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6.1. Cholestasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Liver cirrhosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Cystic liver disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. AQP1 in Cryptosporidium parvum invasion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5. Cholesterol gallstone disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and future perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction In the hepatobiliary tract, water movement is tightly correlated to lipid absorption and secretion, in spite of the poor intrinsic miscibility between the two components. Bile is the ultimate product of hepatobiliary secretory absorptive function, and such aspects are illustrated in the following paragraphs.

2. Biological relevance of membrane water transport Water is the most abundant component of living organisms, and its movement into and out of the living cell is fundamental to life. Maintenance of fluid homeostasis, a housekeeping function of pivotal importance, critically depends on the efficient regulation of water supply and distribution. Living cells have developed complex physiological systems for sensing and responding to changes in fluid composition and volume (Portincasa et al., 2008c). Fluid homeostasis is a highly intricate function that requires a coordinated and precise regulation of solute and water transport systems (Finkelstein, 1987).

3. Aquaporins, highways for cells to recycle water with the outside world Water can cross the biological membranes by simple diffusion, through the lipid bilayer, or by facilitated diffusion, through specialized proteinaceous water channels (Benga et al., 1986a,b; Macey and Farmer, 1970; Finkelstein, 1987). Following the early definition of water channels as aquaporins (AQPs), it became evident that the water permeability of membranes containing AQPs is 5- to 100-times higher than that of membranes lacking such channels (Preston et al., 1992; Agre et al., 1993). Driven by osmotic forces, each single AQP pore can conduct billions of water molecules per second. Testifying their biological importance, AQPs are largely present in nature, being found in microorganisms and in the plant and animal kingdoms (Calamita, 2005). They are small integral proteins consisting of six transmembrane domains connected by five connecting loops (A–E), with molecular weights ranging between 25 and 34 kDa. Connecting loops B and E partially deep the lipid bilayer and each contains the highly conserved signature motif Asn-Pro-Ala (NPA). The so-called aromatic/arginine constriction (ar/R constriction) formed by four defined residues at the extracellular aqueous pore mouth, is of critical importance for the molecular selectivity of the channel. Within the membrane AQPs are organized as tetramer where each monomer contains an individual aqueous pore (Walz et al., 2009). Mammals contain thirteen distinct aquaporins (AQP0–AQP12), functionally subdivided in orthodox aquaporins (AQP0–2, AQP4–6 and AQP8) and aquaglyceroporins (AQP3, AQP7, AQP9 and AQP10) depending on their properties of conducting only water, or glycerol, urea, and some other small neutral solutes, in addition to water, respectively. However, such classification is not clear of blame (Ishibashi et al., 2011). AQP6 is poorly permeable to water while shows conductance to nitrate and other inorganic anions in response to acidic pH or Hg2+ (Yasui et al., 1999). AQP8 features high permeability to ammonia (Jahn et al., 2004; Saparov et al., 2007; Soria et al., 2010) and hydrogen peroxide (Bienert et al., 2007) in addition to water and is often classified as an aqua-ammoniaporin (Jahn et al., 2004). AQP11 and AQP12 are quite divergent compared with the other mammalian AQPs and have been tentatively indicated as unorthodox aquaporins due to their highly variable NPA boxes (Ishibashi et al., 2011). Strong attention is addressed to the roles played by AQPs in bile formation and bile flow, processes characterized by conspicuous movements of water into and out the epithelial cells composing the biliary tree (Portincasa et al., 2008c). Mechanism accounting for molecular transport of water from sinusoidal blood to the bile canaliculus or the bile duct has recently raised much interest. This is due to the recognition of multiple members of the aquaporin (AQPs, aquaporins) family of water channels which are variously expressed in liver epithelial barriers. AQPs appear to be involved in pathways leading to bile formation and hepatic metabolism as well as in hepatobiliary disorders with fluid imbalance (Portincasa et al., 2008c). Here, we summarize and discuss the latest concepts on the nature of bile water transport and review what has been acquired about the aquaporin water channels and their functional implication in bile formation. The current knowledge of clinical relevance of aquaporins in some pathological conditions with abnormal bile flow is also addressed. Emerging areas for future basic and clinical investigation are also addressed.

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4. Hepatic secretion of biliary lipids and bile formation Bile is a complex fluid defined as an aqueous solution of organic and inorganic compounds (Portincasa et al., 2008b). The three lipids bile acids, cholesterol, and phospholipids, together with the bile pigments are the major organic compounds. Proteins have low concentrations in bile and metabolites of various endogenous substances (e.g., hormones) are present at a trace concentration (Portincasa et al., 2008b). Bile is the principal route for the elimination of body cholesterol as unesterified cholesterol or as bile acids. Also, biliary bile acids assist lipid intestinal emulsification and absorption. Lastly, bile permits removal of drugs and toxins from the body. In health, about 0.8–1.0 L of hepatic bile is secreted daily in humans at a rate of 30–40 mL per hour. Canalicular bile is rearranged into the bile duct lumen via secretory and absorptive processes operated by the ductal epithelium, stored and concentrated in the gallbladder, and released into the duodenum (Masyuk and LaRusso, 2006; Muller and Jansen, 1997). Bile water is mostly reabsorbed in the proximal portion of the small intestine (Ma and Verkman, 1999) whereas bile salts are reabsorbed in distal ileum. Reabsorbed bile salts are then taken back to the liver by the enterohepatic circulation (Vlahcevic et al., 1996; Sherlock and Dooley, 2002). Bile formation begins at the level of the bile canaliculus as an osmotic process involving solutes and water. The driving force necessary to bile formation is the active concentration of bile acids and other biliary constituents in the bile canaliculi. Solutes include three classes of biliary lipids which are actively transported, i.e., cholesterol (transported into the canaliculus by ABCG5 and ABCG8, two plasma membrane proteins ATP-binding cassette (ABC) transporters), phospholipid (transported by ABCB4), and bile acids (transported by ABCB11) (Portincasa et al., 2006b). Bile acids represent the main organic solutes driving bile formation. Total lipid concentration of bile is 3 g/dL (3% of hepatic bile by weight) with concentrations set at 20–30 mM (12 g/L) for bile acids, 7 mM (5 g/L) for phospholipid, and 2–3 mM (1 g/L) for cholesterol. Bile acids enter canalicular spaces as monomers while biliary phospholipids and cholesterol are secreted into bile as unilamellar vesicles (40–120 nm radium) (Portincasa et al., 2008b). Transport systems involved in hepatic secretion of biliary lipids in the human liver with their corresponding functions are depicted in bile, formed by the hepatocytes, is secreted in the bile canaliculi and then modified during its passage in the bile ductules and ducts. Since canalicular bile flow can also be found in the absence of bile acids or at low bile acid outputs, two components for canalicular bile formation have to be described. The first component is the bile acid-dependent bile flow (i.e., bile flow related to bile acid secretion). In this case, increased bile acid secretion promotes bile flow because bile acids provide a major osmotic driving force for filtration of water and electrolytes. The second component is the bile acid-independent bile flow (i.e., bile flow attributed to active secretion of osmotically active inorganic electrolytes and organic anions) which accounts in humans for 1.5–2.0 L/ min/kg/body weight. Na+, K+, Ca++, Mg++, Cl, and HCO3- are the major inorganic electrolytes which, in the common duct bile have concentrations very close to those observed in plasma. Hepatic secretion of the organic anion glutathione (GSH) is ATP-dependent and depends strictly on the multidrug resistance-associated protein 2 (MRP2) placed on the canalicular   membrane of hepatocyte. Hepatic secretion of bicarbonate ðHCO anion exchanger AE2 and oc3 Þ requires the HCO3 =Cl curs at the level of the bile duct epithelial cells in response to stimulation by a variety of hormones and neuropeptides, such as secretion and vasoactive intestinal peptide. Both GSH and HCO3- secretion represent the major components of the bile acid-independent fraction of bile flow. Intraluminal catabolism of GSH by glutamyl transpeptidase (GGTP) also contributes to the osmotic driving force for canalicular bile formation. Because of the activity of the membrane-bound enzyme Na+, K+-ATPase, active sodium transport into the canaliculi also induces bile acid-independent bile flow (Wang et al., 2009). Lastly, total bile flow consists of constant ductal/ductural secretion and total canalicular bile flow. The relation is linear in both total bile flow and total canalicular bile flow. Unconjugated dihydroxy bile acids secreted into bile are passively absorbed by cholangiocytes, returned to the hepatocyte via the periductular capillary plexus, and re-secreted into bile, depicting the so-called ‘‘cholehepatic shunt’’. Absorption of the protonated unconjugated bile acid molecule generates a bicarbonate anion, resulting in a bicarbonate-rich choleresis. Premature absorption and resecretion of the bile acid also induce bile acid-dependent bile flow. The role of this shunt appears to be minor and possibly related to a role of cholangiocytes to ‘‘sample’’ biliary bile acid concentrations and activate cellular signaling pathways, rather than to transport significant quantities of bile acids. What is fascinating in scenario, is that bile is an aqueous solution transporting high concentrations of solubilized cholesterol which is virtually insoluble in water (Wang et al., 2008). A complex pathway involving lipidic carriers of cholesterol is involved to facilitate cholesterol transport. Since lecithins (phospholipids) are also insoluble in water, the third lipid component, bile acids (amphiphilic molecules), are essential for transporting biliary cholesterol by forming macromolecular aggregates in bile, namely micelles and vesicles. Bile acid monomers can aggregate spontaneously to form simple micelles if the critical micellar concentration (CMC) is exceeded (about 2 mmol/L). Simple micelles (3 nm in diameter) are small, thermodynamically stable aggregates that solubilize little cholesterol. When simple micelles start incorporating phospholipids, mixed larger micelles form (4–8 nm in diameter), in which three times more cholesterol is solubilized. Mixed micelles develop as lipid bilayer with the hydrophilic groups of the bile acids and phospholipids exposed on the outside of the bilayer and the hydrophobic groups on the inside. Maximal solubility of cholesterol occurs when the molar ratio of phospholipids to bile acids is 0.2–0.3. Of note, gallbladder bile from many healthy individuals is supersaturated with cholesterol, indicating that cholesterol concentrations exceed what could be solubilized by micellar particles. A role in this case is essential for vesicles (40–100 nm in diameter): unilamellar spherical structures contain-

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Table 1 Bile acid and organic solute transporters involved in bile formation at the hepatocyte and cholangiocyte plasma membrane. Transporter abbreviation

Name

Gene

Location

Function

ABCG5/G8a

Sterolin-1 and -2

BRCPa

Breast cancer resistance protein

ABCG5/ G8 ABCG2

APM and apical membrane of enterocytes APM and proximal tubule of kidney

BSEPa

Bile salt export pump

ABCB11

APM

MATE-1

Multidrug and toxin extrusion protein 1 Multidrug-resistance-1 Pglycoprotein

SLC47A1

APM and kidney brush border APM and cholangiocyte apical membrane

Multidrug-resistance-3 Pglycoprotein (phospholipid transporter) Multidrug-resistance-associated protein 2 (canalicular multispecific organic anion transporter) Multidrug-resistance- associated protein 3 Multidrug-resistance-associated protein 4

ABCB4

APM

ATP-binding cassette G5 and G8. Heteromeric ATPdependent transport for cholesterol and plant sterols ATP-dependent multispecific drug transporter, particularly sulfate conjugates; protoporphyrins are endogenous substrate. Substrate overlap with MRP2 ATP-dependent BA transport into bile; stimulate bile salt-dependent bile flow Organic cation/H + exchanger extrudes cationic xenobiotics ATP-dependent excretion of various organic cations, xenobiotics, and cytotoxins into bile; barrier function in cholangiocytes ATP-dependent translocation of phosphatidylcholine from inner to outer leaflet of membrane bilayer

ABCC2

APM

ABCC3

MRP6a

Multidrug-resistance- associated protein 6

ABCC6

BLPM of hepatocytes and cholangiocytes BLPM of hepatocytes and apical membrane of proximal tubule of kidney BLPM of hepatocytes

NPC1L1

Niemann-Pick C1-like 1 protein

NPC1L1

NTCP

Sodium-taurocholate cotransporting polypeptide Orgenic anion-transporting polypeptides

SLC10A1

MDR1a

MDR3a

MRP2a

MRP3a MRP4a

OATPs

ABCB1

ABCC4

OAT-2

Organic anion transporter 2

SLCO1B1 and SLCO1B 3 SLC22A7

OCT-1

Organic cation transporter-1

SLC22A1

OST a/b

Organic solute transporter a/b

APM and apical membrane of enterocytes BLPM of hepatocytes BLPM of hepatocytes

BLPM of hepatocytes

BLPM of hepatocytes

BLPM of hepatocytes, cholangiocytes, ileum and proximal tubule of kidney

Mediates ATP-dependent multispecific organic anion transport (e.g., bilirubin diglucuronide) into bile; contributes to BA-independent bile flow by GSH transport Expression induced in cholestasis. Transports bilirubin and BA glcuronide conjugates Expression induced in cholestasis-transports sulfated BA conjugates and cyclic nucleotides ATP-dependent bile salt transport of organic anions and small peptides. Mutations of MRP6 gene result in pseudoxanthoma elasticum Sterol import in the liver (in humans) and small intestine (humans, rodents) Primary carrier for conjugated BA uptake from portal blood Broad substrate carriers for sodium-independent uptake of BA, organic anions, and other amphipathic organic solutes from portal blood Facilitates sodium-independent hepatic uptake of drugs and prostaglandins Facilitates sodium-independent hepatic uptake of small organic cations Heteromeric solute carrier for facilitated transport of BA across basolateral membrane of ileum. Expression induced in liver in cholestasis

Abbreviations: APM, apical plasma membrane (canalicular membrane); BA, bile acids; BLPM, basolateral plasma membrane. a Member of the superfamily of ATP-binding cassette (ABC) transporters; Adapted from Boyle (2009) and Dawson et al. (2009).

ing cholesterol, phospholipids, and little bile acids. Vesicles are likely secreted by hepatocytes (Crawford et al., 1995) and are larger than both simple and mixed micelles. It is believed that vesicle is stable particle responsible for solubilizing biliary cholesterol in excess of what could be solubilized in mixed micelles. The relative ratio of three biliary lipids is influenced by the fasting or the feeding states through alterations in hepatic secretion rates. This process is inevitably influencing the interaction across biliary particles as the flow from the secretory pole of the hepatocyte towards the biliary tract, and the gallbladder, where bile is further concentrated. In the fasting state biliary bile acid output is relatively low, the ratio of cholesterol to bile acids is increased and cholesterol is principally carried in vesicles rather than in micelles. During meals, biliary bile acid output is higher and more cholesterol appears in micelles. Dilute bile holds stable vesicles with low conversion rate to micelles. The opposite occurs with increasing bile acid concentrations in concentrated gallbladder bile. Because relatively more phospholipids than cholesterol can be transferred from vesicles to mixed micelles, the residual vesicles are remodeled, which may be enriched in cholesterol relative to phospholipids. When cholesterol/phospholipid ratio in vesicles is greater than 1, vesicles become increasingly unstable. Thus, unilamellar vesicles can aggregate and fuse to form large multilamellar vesicles (also known as liposomes or liquid crystals, 500 nm in diameter) that are composed of multilamellar spherical structures. Notably, the seminal step in the pathogenesis of cholesterol gallstones include formation of solid plate-like cholesterol monohydrate crystals nucleating from multilamellar vesicles in concentrated gallbladder bile (Portincasa et al., 2006a,b; Wang et al., 2009; Krawczyk et al., 2011; de Bari et al., 2012; Wang et al., 2010).

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P. Portincasa, G. Calamita / Molecular Aspects of Medicine 33 (2012) 651–664 Table 2 Reported localization and suggested physiological and pathophysiological relevance of hepatobiliary AQPs. Hepatobiliary compartment

Aquaporin

Cellular location

Subcellular location

Suggested functional involvement

Suggested clinical relevance

Liver

AQP8

Hepatocytes

APM; SAV; ER; IMM

Cholestasis

AQP9

Hepatocytes

BLPM

AQP11

Hepatocytes

ER?

Secretion of canalicular bile water; preservation of cytoplasm osmolarity during glycogen synthesis and degradation; mitochondrial ammonia detoxification and ureagenesis; mitochondrial ROS generation Import of water from sinusoidal blood; uptake of glycerol during starvation; maintenance of hepatocellular hydration state; urea efflux (protein catabolism) Undefined

AQP3 AQP1

Hepatocytes Cholangiocytes

Undefined APM; SAV

Undefined Secretion and absorption of ductal bile water

AQP4 AQP1

Cholangiocytes Epithelial cells

Secretion and absorption of ductal bile water Cystic bile absorption/secretion (?)

AQP8

Epithelial cells

AQP4

Epithelial cells (?)

BLPM APM; SAV; BLPM APM; IC(weak) Undefined

Endothelia

AQP1

APM; BLPM

Bile formation and flow

Hepatic stellate cells

Multiple AQPs

Portal sinusoids; peribiliary vascular plexus; blood vessels Stellate cells

Cystic liver disease Undefined Biliary cryptosporidiosis Undefined Cholesterol gallstone disease Cholesterol gallstone disease Gallstones associated with obesity Liver cirrhosis

Undefined

Activation of hepatic stellate cells

Undefined

Intrahepatic bile ducts Gallbladder

Cystic bile absorption (?) Cystic bile absorption/secretion (?)

Cholestasis; metabolic syndrome

APM, apical plasma membrane; BLPM, basolateral plasma membrane; ER, endoplasmic reticulum; SER, smooth endoplasmic reticulum; SAV, subapical plasma membrane; IC, intracellular location.

5. Distribution, regulation and physiology of biliary aquaporins in mammals The epithelial cells of the mammalian hepatobiliary tract express at least six distinct aquaporins (AQP1, 3, 4, 8, 9, and 11) variously distributed among the different system sections (Table 2). AQPs are also present in hepatic stellate cells (Jia et al., 2011; Lakner et al., 2011; Yovchev et al., 2008). 5.1. Hepatocyte express multiple aquaporins Rodent hepatocytes express AQP8, 9 and 11 (Elkjaer et al., 2000; Nihei et al., 2001; Calamita et al., 2001; Morishita et al., 2005). A fourth AQP, AQP3, is found in human hepatocytes (Ishibashi et al., 1995). The distinctive subcellular localization characterizing these AQPs may justify their redundancy. 5.1.1. AQP8 Aquaporin-8, the most abundant AQP in hepatocytes, is marked by a divergent evolutionary pathway and unusual genomic organization when compared to the other mammalian AQPs (Calamita et al., 1999; Ferri et al., 2003; Zardoya, 2005). Two AQP8 isoforms with distinctions in their N-termini, generated by alternatively splicing have been found in rodent hepatocytes (Portincasa et al., 2003; Calamita et al., 2005b). AQP8 features multiple subcellular localization being found at the canalicular plasma membrane and in subapical vesicles (Garcia et al., 2001; Calamita et al., 2001; Ferri et al., 2003), inner membrane of mitochondria (Ferri et al., 2003; Calamita et al., 2005b) and membranes of smooth endoplasmic reticulum (SER) adjacent to glycogen granules (Ferri et al., 2003). Functional studies suggest apical AQP8 as the channel that mediates the canalicular secretion of water in hepatocyte bile formation (Huebert et al., 2002; Marinelli et al., 2003; Larocca et al., 2009). This is in line with an ontogenetic study with rats showing that both the mRNA and protein expression of liver AQP8 increase abruptly at the time of weaning when the hepatobiliary transport systems complete their maturation (Ferri et al., 2003), as well as with a series of works using different models of cholestasis where defective hepatic AQP8 expression was shown to contribute to the induced bile secretory dysfunction (Carreras et al., 2003, 2007; Lehmann et al., 2008a). A flow chart describing the functional involvement of AQP8 in primary bile formation is described in Fig. 1. Following stimulation by choleretic agonists, such as dibutyryl cyclic adenosine monophosphate (cAMP) or glucagon, subapical AQP8 translocates to the canalicular plasma membrane. This redistribution increases the water permeability of the apical plasma membrane leading to facilitation of the osmotically driven water transport into the lumen of the bile canaliculus (Garcia et al., 2001; Huebert et al., 2002; Gradilone et al., 2003). This working mod-

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el was extended by studies showing that, like other canalicular transporters mediating primary bile secretion, hepatocyte AQP8 is significantly and specifically increased in apical lipid ‘‘raft’’ microdomains involved in canalicular secretion, following exposure to the choleretic agonist glucagon (Tietz et al., 2005; Mazzone et al., 2006). The cAMP/glucagon-induced redistribution of AQP8 to the canalicular plasma membrane was suggested to occur via phosphatidylinositol-3-kinase-dependent microtubule-associated trafficking of vesicles (Gradilone et al., 2005). This is also the case of carriers involved in canalicular  bile secretion such as the isoform 2 of the Cl =HCO 3 exchanger (AE2) and the multidrug resistance-associated protein 2 (MRP2). This scenario is consistent with a study in rat primary hepatocytes, glucagon showing increased expression of AQP8 due to a reduction of its degradation by a process involving cAMP-PKA and PI3K signal pathways (Soria et al., 2009). However, hepatocytes from AQP8 knockout mice were reported to have similar water permeability to those from wild-type mice (Yang et al., 2005). This apparent discrepancy may come from the redundancy characterizing AQPs in hepatocyte and the functional modification of other genes in response to the elimination of the target gene as well as from the fact that that a 60% reduction of AQP8 protein in the rat hepatocyte apical membrane corresponds to a 15% decrease in the overall canalicular osmotic permeability (Carreras et al., 2007). The extent of expression of AQP8 among rodent mitochondria is variable with heavy mitochondria showing the higher levels of immunoreactivity. Surprisingly, in spite of its high conductance to water, AQP8 did not appear to have major relevance in facilitating the movement of water into and out the mitochondrial matrix (Calamita et al., 2006; Gena et al., 2009). Since rat, mouse, and human AQP8 are able to efficiently mediate the transport of ammonia (Jahn et al., 2004; Liu et al., 2006;

Fig. 1. The scenario of bile formation is depicted in hepatocytes, with respect to hepatobiliary transporters and solutes. For clarity reasons, transporters are reported in different parts of the panel, although they all operate within the same cell. Left hepatocyte: at the sinusoidal domain of the basolateral plasma membrane of hepatocytes, two transport systems operate: OATP (namely OATP1B1: SLCO1B1 and OATP1B3: SLCO1B3) for uptake of unconjugated BA, OA, OC and efflux of GSH and bicarbonate) and NTCP (SLC10A1) for sodium-dependent uptake of BA. NTCP function requires the Na+/K+-ATPase generating an inwardly directed Na+ gradient and a K+ channel (which generates in part the membrane potential). Right hepatocyte: at the domain of the basolateral membrane additional transporters include a Na+-bicarbonate cotransporter (symporter), a Na+–H+ exchanger; MRP3, MRP4, and OSTa/b all three contribute to BA, OA, and solute transport. At the canalicular membrane (apical plasma membrane) of hepatocytes, three ATP-binding cassette (ABC) transporters operate: ABCB4 (also named Multidrug-resistance-3 P-glycoprotein MDR3) for phospholipid (PL), ABCB11 (also named Bile salt export pump BSEP) for bile acid (BA), and ABCG5/G8 for cholesterol (Chol) secretion which concur to formation of micelles and vesicles in bile (see text for further details). The NPC1L1 protein is responsible for sterol import and is found in the liver in humans but is absent in rodents (Pramfalk et al., 2011). Drug metabolites are secreted by the MDR1. Sulphated (BA-S) or glucuronidated (BA-G-) bile acids, and OA are secreted by the MRP2. Canalicular membrane-associated ATP-independent transport systems include the GSH transporter, the AE2 for secretion of bicarbonate, and a chloride channel (distinct from the cystic fibrosis transmembrane regulator protein, CFTR). Canalicular formation and secretion of water in bile are depicted in the right hepatocyte. Water flows basolaterally from the sinusoidal blood into the hepatocyte through AQP9. Under choleretic stimuli, an increase of intracellular cAMP, subapical vesicles containing AQP8 are translocated by exocytosis to the canalicular membrane. Here, AQP8 facilitates the osmotically-driven efflux of water in the bile canaliculus. AQP8 is also present in mitochondria (inner membrane) and in the smooth endoplasmic reticulum, where it is suggested to play other functions unrelated to bile formation (not shown). Abbreviations: AE2, chloride–bicarbonate anion exchanger isoform 2; AQP8, aquaporin-8; AQP9, aquaporin-9; BA, bile acids; BA-G, glucuronidated bile acids; BA-S, sulphated bile acids; Chol, cholesterol; GSH, glutathione; MDR1, Multidrug-resistance-1 P-glycoprotein; MRP2, Multidrug resistance-associated protein 2; MRP3, Multidrug resistance-associated protein 3; MRP4, Multidrug resistance-associated protein 4; NCTP, sodiumtaurocholate cotransporting polypeptide; NPC1L1, Niemann-Pick C1 Like 1 protein; OA, organic anion; OATP, sodium-independent organic-anion transporting polypeptide; OC, organic cation; OS, organic solutes; OSTa/b, Organic solute transporter a/b; PL, phospholipids. See also Tables 1 and 2 for abbreviations. Adapted from Dawson (2010), Portincasa et al. (2008b), Takeyama and Sakisaka (2011), and Calamita et al. (2005c).

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Holm et al., 2005; Saparov et al., 2007), facilitation of ammonia membrane transport might be an important function for AQP8 in hepatocyte mitochondria (Soria et al., 2010). Thus, it is reasonable to suppose that the AQP8 may play a role in the hepatic mitochondrial ammonia detoxification and ureagenesis (urea cycle). AQP8 has been also suggested to facilitate the release of H2O2 from the mitochondrial matrix during generation of reactive oxygen species (Bienert et al., 2007). In rat liver, mitochondrial AQP8 is modulated negatively by triiodothyronine (Calamita et al., 2007), a modulation that may be relevant to the regulation of mitochondrial metabolism by thyroid hormones. However, the precise functional significance of AQP8 in hepatocyte mitochondria is object of current investigation. Smooth endoplasmic reticulum AQP8 is speculated to mediate the osmotic movement of water between SER lumen and the region of the hepatocyte cytoplasm where newly deposited glycogen occurs and, therefore, have a role in preserving cytoplasmic osmolality during glycogen synthesis and degradation in the liver (Ferri et al., 2003). 5.1.2. AQP9 Aquaporin-9 is an aquaglyceroporin of broad selectivity that in liver parenchyma localizes exclusively at the sinusoidal domain of the hepatocyte basolateral plasma membrane (Elkjaer et al., 2000). While apparently unaffected by the choleretic stimulus (Huebert et al., 2002; Marinelli et al., 2003), AQP9 is negatively regulated by insulin (Kuriyama et al., 2002; Carbrey et al., 2003) and leptin (Rodriguez et al., 2011b) at gene level. AQP9 is upregulated by fasting in wild type mice but not in mice lacking the c isoform of the peroxisome proliferator-activated receptor (PPARc) (Patsouris et al., 2004). The metabolic relevance of AQP9 is object of a body investigation (Rojek et al., 2007; Maeda et al., 2008; Rodriguez et al., 2011a). The longstanding postulation making AQP9 the primary entry pathway by which plasma glycerol, the major gluconeogenetic substrate during fasting, is imported by hepatocytes has been recently proven experimentally (Calamita et al., 2012). AQP9 underlies the fluctuation of liver glycerol permeability depending to the nutritional status and circulating levels of insulin. Recently, sexual dimorphism in the hepatic handling of glycerol during starvation associated with 17b-estradiol prevention of the fasting-induced increase of liver AQP9 has been also observed (Lebeck et al., 2012). Hepatocyte AQP9 does not merely represent a pathway for the import of glycerol since it is also suggested to facilitate the osmotic uptake of water from the sinusoidal blood into the rat hepatocyte cell interior (Calamita et al., 2008). As depicted in Fig. 1, together with AQP8, AQP9 contributes to primary bile formation and secretion (Portincasa et al., 2008c). Like AQP8, defective AQP9 expression and reduced basolateral osmotic water permeability have been reported to be associated to secretory dysfunction of rat hepatocytes during obstructive extrahepatic cholestasis (Calamita et al., 2008). AQP9 may be also important for the rapid shifts of water across, into, or out of the hepatocyte underlying the hepatocellular hydration state, an efficient mechanism of short-term control of canalicular secretion and hepatocyte volume (Haussinger and Schliess, 1999; Haussinger et al., 2000). A role for AQP9 as exit channel for urea produced within the hepatocyte or solutes, such as purines and pyrimidines derived from nucleotide synthesis de novo, lactate and ketone bodies has been also hypothesized (Tsukaguchi et al., 1998). Based on its proven capacity to transport certain heavy metals, AQP9 is speculated to represent the entry route of arsenic in hepatocyte (Liu et al., 2002) whose consequent poisoning is known to lead to hepatocellular damage and hepatocellular carcinoma. Novel drugs have been recently identified that specifically inhibit AQP9 (Jelen et al., 2011) by opening an interesting chapter in the targeting of such aquaporin channel for therapeutical purposes. 5.1.3. AQP11 Aquaporin-11 is an unusual AQP marked by its deviated pore-forming NPA boxes (Ishibashi et al., 2011), intracellular localization and weak conductance to water (Yakata et al., 2011). In mouse, in addition to hepatocytes, AQP11 is expressed in a series of other tissues (Morishita et al., 2005). Interestingly, AQP11 null mice show vacuolization in hepatocytes of the portal area suggesting involvement of such AQP in hepatic fluid homeostasis. The AQP11/ mice die before weaning as a consequence of the uremia from polycystic kidney disease (Morishita et al., 2005). The functional significance of AQP11 is object of intense debate and investigation (Ishibashi et al., 2011; Isokpehi et al., 2009) and light will be provided in the next future. 5.1.4. AQP3 Aquaporin-3 mRNA is found in human hepatocytes (Ishibashi et al., 1995). However, the presence of the corresponding protein and related functional relevance in liver remains elusive. Further investigation is needed to fully assess both the subcellular localization and functional meaning of AQP3 in liver. 5.2. Aquaporins of the intrahepatic bile ducts Cholangiocytes, the epithelial cells lining the intrahepatic bile ducts, play an important role in bile formation by contributing up to 40% of the daily output of bile fluid to form the so called ductal bile (Marinelli et al., 1997, 1999). At a protein level, rat cholangiocytes express AQP1 and AQP4, whose regulation and functional involvement has been object of intense investigation (see (Masyuk and LaRusso, 2006) for a review) (Fig. 2). In basal conditions, cholangiocyte AQP1 was reported to reside in the membrane of subapical vesicles whereas under active choleresis secretin would stimulate the exocytotic insertion of AQP1 in the apical plasma membrane facilitating the secretion of water into the biliary lumen (Tietz et al., 2003; Splinter et al., 2004). In such working model, at the basolateral side of cholangiocytes, AQP4 would mediate the import of

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Fig. 2. The scenario of bile formation is depicted in intrahepatic bile duct cholangiocytes, with respect to hepatobiliary transporters and solutes. For clarity reasons, transporters are depicted in different parts of the panel, although they all belong to the same cell. Left cholangiocytes: proposed model for the coupling of solute and water transport and somatostatin-induced absorption of ductal bile in rat cholangiocyte. Upper cholangiocyte: main transporters involved in bile duct secretion. The increase in intracellular cAMP induced by choleretic hormones such as secretin activates CFTR leading to the exocytotic insertion of AQP1 in the cholangiocyte apical membrane. The efflux of Cl- leads to the extrusion of HCO3- (via the Cl-/HCO3- exchanger, AE2) and Na+ (paracellular arrow) and possibly K+ (through a paracellular pathway; not depicted in the figure). The osmotic gradient created by these solute transports drives a transcellular movement of water into the bile duct lumen. The existence of a subapical vesicles containing transporters involved in bile duct secretion including CFTR, AE2 and AQP1 has been recently suggested in rat cholangiocytes (Tietz et al., 2003). This vesicles translocate exocytotically into the apical membrane under stimulation of secretin. Water is imported mostly by AQP4 and secreted into the lumen by AQP1. Lower cholangiocyte: main transporters involved in bile duct absorption. Somatostatin decreases the ductal bile secretion by reducing the intracellular levels of cAMP and, consequently, inhibiting the exocytotic insertion of AQP1 and the activation of CFTR in the apical membrane. The net water ductal absorption caused by somatostatin is the consequence of stimulating glucose and bile salt absorption by SGLT1 and ASBT, respectively. The sodium/hydrogen exchanger isoform NHE3 has also been suggested to be involved in bile duct secretion. Of note, in this working model, based on bidirectional property, AQP4 would move water into or out of the cholangiocyte, depending on the secretive or absorptive status of the bile duct. Right cholangiocytes: at the apical membrane, glucose and amino acids are reabsorbed by the GLUT1 and amino acid carriers. On the basolateral membrane bile acids may exit by the MRP3 and the heteromeric Organic solute transporters OSTa/b Abbreviations: ASBT, apical Na+-coupled bile salt transporter; AQP1, aquaporin-1; AE2, Cl-/HCO3- anion exchanger; AQP4, aquaporin-4; BA, bile acids; BA-G, glucuronidated bile acids; BA-S, sulphated bile acids; CFTR, cystic fibrosis transmembrane conductance regulator Cl- channel; Chol, cholesterol; GLUT1, glucose transpoter isoform 1; MRP3, Multidrug resistance-associated protein 3; NHE3, Na+/H+ exchanger isoform 3; OA, organic anion; OC, organic cation; OS, organic solutes; SGLT1, Na+-coupled glucose transporter. See also Tables 1 and 2 for abbreviations. Adapted from Dawson (2010), Portincasa et al. (2008b), and Takeyama and Sakisaka (2011).

water from the peribiliary vascular plexus surrounding the bile duct (Masyuk et al., 2000). Masyuk and coworkers suggested that both AQP1 and AQP4 are also involved in the intrahepatic bile duct absorption of water (Masyuk et al., 2002b). Most of the driving force underlying the ductal absorption of water would be generated by the uptake of conjugated bile acids (cholehepatic circulation of bile acids) and glucose by the apical Na+-dependent bile acid transporter ASBT and Na+-coupled glucose transporter SGLT1 (Ricci and Fevery, 1981; Tietz et al., 1995). Reabsorption of glucose and/or bile acids by cholangiocytes would be stimulated by somatostatin (Mennone et al., 2002), a hormone with cholestatic action. However, ultimate validation of the suggested mechanisms involving AQPs in rat bile duct formation requires additional studies, especially in the light of observations that in mice AQP1 is not regulated by cAMP and that its gene deletion does not apparently affect bile formation (Mennone et al., 2002). Work needs to be addressed to evaluate the presence at protein level and physiological relevance of AQP11 in cholangiocytes. 5.3. Gallbladder aquaporins The epithelial cells of human and mouse gallbladder express AQP1 and AQP8 (van Erpecum et al., 2006; Nielsen et al., 1993; Calamita et al., 2005a) (Li et al., 2009). In the human gallbladder, AQP1 is localized on both the apical and basolateral

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plasma membranes of epithelial cells lining the neck portion of the organ (Calamita et al., 2005a). In the mouse gallbladder, AQP1 is localized at the apical and basolateral plasma membranes as well as in subapical vesicles of the epithelial cells lining the neck and corpus portions (van Erpecum et al., 2006). The mRNA expression of mouse gallbladder AQP1 is slightly upregulated by the satiety hormone leptin (Swartz-Basile et al., 2007). In the mouse gallbladder epithelium, AQP8 is predominantly localized at the plasma membrane and, at lesser extent, intracellularly. AQP8 was also detected in rabbit, calf and guinea pig gallbladders (Calamita et al., 2005a). This pattern of subcellular localization makes reasonable to speculate that AQP8 and AQP1 facilitates the transport of water through the apical membrane whereas basolateral AQP1 mediates the movement of water at the serosal side of the epithelial cells. The presence of two distinct AQPs at the apical plasma membrane may relate to the dual function exerted by the luminal membrane, namely the absorbtion and secretion of fluid from and into the gallbladder lumen. AQP1 might be (hormonally?) translocated to the apical membrane to secrete water as in the bile duct epithelium, a functional homolog of the gallbladder epithelium, whereas apical AQP8 might account for the absorption of water from gallbladder bile. Very high transepithelial osmotic water permeability was recently reported by the Verkman group in intact mouse gallbladder resulting from transcellular water movement through plasma membrane AQP1 water channels (Li et al., 2009). The presence of AQP8 water channels at the apical pole of the epithelium was also confirmed. Based on the obtained results, the same Authors argued that: (i) the high, AQP1-facilitated water permeability is constitutive rather than cAMP-regulated; (ii) while with a role in water permeability, AQP1 is not physiologically important in gallbladder bile reworking and; (iii) despite the small increase in AQP8 transcript expression found in AQP1 null gallbladders, AQP8 does not functionally substitute for AQP1 in the AQP1 null gallbladders. These conclusions do not fit with a study with mice fed a lithogenic diet showing temporal association between decreased gallbladder concentrating function and reduced AQP1 or AQP8 expression and suggesting the involvement of these water channels in gallbladder water transport (van Erpecum et al., 2006). Involvement of AQPs in mouse cystic bile physiology was also suggested in a study using leptin-deficient animals where leptin replacement moderately enhanced gallbladder AQP1 whereas downregulated AQP4. Leptin was hypothesized to alter gallbladder volume by mediating gallbladder absorption/secretion of water acting on the expression of AQPs (Goldblatt et al., 2002). While this apparent discrepancy may be dispelled by a careful evaluation of the experimental conditions with which mice were analyzed further work is urgently warranted in this field. 5.4. AQPs of hepatobiliary endothelial cells Consisting with its general expression in endothelial cells, AQP1 is found in the hepatic and gallbladder blood vessels (Talbot et al., 2003). In particular, its high expression in the peribiliary vascular plexus, a mesh-like arrangement of blood vessels that surround the intrahepatic bile ducts indicates a functional role in mediating water transport from plasma to bile (Masyuk et al., 2002a). 6. Pathophysiology of biliary aquaporins The relevance of AQPs in both health and disease in humans is a growing new area of investigation, with key diagnostic and therapeutic implications (King et al., 2004; Verkman, 2012). A number of diseases affecting the hepatobiliary system have been associated with abnormal fluid transport and consequent cholestasis (Zollner and Trauner, 2008). Dysregulation of hepatobiliary AQPs and bile secretion impairment has been observed in experimental models of cholestasis such as extrahepatic, estrogen-induced and sepsis-induced cholestases. Studies with mouse models of gallstone disease showed association between decreased gallbladder AQP expression and gallbladder concentrating function (van Erpecum et al., 2006). Clinical relevance has been also suggested for AQPs in liver cirrhosis, polycystic liver and biliary cryptosporidiosis. 6.1. Cholestasis Studies in several experimental models of cholestasis including extrahepatic obstructive cholestasis (Carreras et al., 2003), estrogen-induced cholestasis (Carreras et al., 2007), and sepsis-induced cholestasis (Lehmann and Marinelli, 2009), have suggested that defective canalicular AQP8 expression contributes to the development of cholestasis (see Marinelli et al., 2012 for an update review). The cholestatic relevance of AQP8 was also suggested by biophysical experiments with canalicular plasma membranes revealing decreased canalicular water permeability associated with AQP8 protein downregulation (Carreras et al., 2003, 2007). Marinelli and coworkers suggested that the combined alteration in hepatocyte solute transporters and AQP8 hamper the efficient coupling of osmotic gradients and canalicular water flow and, therefore, cholestasis may result from a mutual occurrence of impaired solute transport and decreased water permeability (Lehmann et al., 2008b). In line with such hypothesis, AQP9, the basolateral AQP suggested to contribute to the transport of water from the sinusoidal blood into hepatocyte, was found to be downregulated post-transcriptionally in a rat model of extrahepatic cholestasis (Calamita et al., 2008). 6.2. Liver cirrhosis Cirrhosis is the end stage of progressive hepatic fibrosis which is characterized by distortion of the hepatic architecture and the formation of regenerative nodules. In its advanced stage, cirrhosis is irreversible and the only therapeutic option

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remains liver transplantation (Sherlock and Dooley, 2002). A recent study in normal and cirrhotic liver tissues derived from human and mouse found that AQP1 enhances osmotic water permeability and FGF-induced dynamic membrane blebbing in liver endothelial cells. This mechanism is mediated by driving invasion and pathological angiogenesis during cirrhosis (Huebert et al., 2010). Aberrant expressions of AQP1 in periportal sinusoidal regions in human cirrhotic liver have been recently suggested contribute to microvascular resistance in cirrhosis (Yokomori et al., 2011). Increased levels of plasma vasopressin and the consequent increase of water absorption by the renal collecting duct, represent an important pathophysiological mechanism underlying the water retention associated with hepatic cirrhosis (Bichet et al., 1982; Nielsen et al., 2007). Both the mRNA and protein levels of AQP2, the vasopressin-induced water channel of the kidney collecting-duct principal cells, resulted upregulated in rats developing carbon tetrachloride-induced liver cirrhosis (Asahina et al., 1995). Treatment with antagonists of V2, the vasopressin receptor, normalize water excretion in rats with cirrhosis and ascites (Claria et al., 1989). However, the transcriptional expression of AQP2 appeared to vary considerably depending on the models of hepatic cirrhosis. In this context, renal AQP2 was found to be downregulated in rats with cirrhosis induced by common duct bile ligation (Fernandez-Llama et al., 2000), and in patients with cirrhosis and ascites (Esteva-Font et al., 2006). An adaptive response was evoked to explain the observed sodium retention, with expansion of extracellular fluid volume and dilutional hyponatremia (Esteva-Font et al., 2006).

6.3. Cystic liver disease Hepatic cystogenesis associated with abnormal expression and subcellular localization of AQP1 (together with the chloride channel CFTR and the anion exchanger AE2), has been revealed in a rat model of autosomal recessive polycystic kidney disease. Liver cysts were hypothesized to be due to increased fluid accumulation following overexpression and abnormal location of AQP1, CFTR, and AE2 in cystic cholangiocytes (Banales et al., 2008). Disruption of the AQP11 gene in mice results in intracellular vacuolization of periportal hepatocytes. Such mice develop a severe form of polycystic kidney disease (PKD) causing uremic death before weaning as a consequence of renal failure (Morishita et al., 2005). Since the life span of AQP11-/mice was limited by the kidney disease where cysts were generated starting from intracellular vacuolization of proximal tubular cells, the liver phenotype might be premature. Polycystic livers are expectable in AQP11 knockout mice by considering that cysts are often observed in the biliary epithelia of PKD patients and mice (Chen et al., 2005). However, work is warranted to verify whether the PKD caused by lack of AQP11 in mice displays the same liver cysts induced by the autosomal recessive form of PKD, a well characterized form of polycystic kidney disease caused by homologous Cpk gene.

6.4. AQP1 in Cryptosporidium parvum invasion A role for AQP1 in mechanisms of invasion of cholangiocytes by the intracellular parasite C. parvum was recently reported by LaRusso and coworkers (Chen et al., 2005). C. parvum infects a number of epithelial cells, including cholangiocytes, and this invasion process is characterized by host–cell membrane protrusion to encapsulate the parasite. In murine cholangiocytes, C. parvum recruits AQP1 and the Na-dependent glucose transporter SGLT1 to the attachment site, generating a localized glucose-driven AQP1-mediated water influx, thereby facilitating the localized host–cell membrane protrusion required for membrane extension and parasitic cellular internalization (O’Hara et al., 2010). Identification and characterization of host molecules required for C. parvum internalization not only may identify novel therapeutic targets for cryptosporidiosis but will provide insight into dynamic cholangiocyte apical-membrane remodeling in general.

6.5. Cholesterol gallstone disease Gallstone disease is one of the most prevalent and costly digestive diseases in Western countries, with a 10–15% prevalence in adulthood (Portincasa et al., 2006a; Sandler et al., 2002). About 80% of the gallstones are cholesterol gallstones (Wang et al., 2009), while the remaining are pigment stones that contain less than 30% cholesterol. The prevalence of gallstones increases with age, while being associated with multiple risk factors (Portincasa et al., 2006a; Katsika et al., 2005). Cholesterol gallstone disease implies disturbed cholesterol homeostasis leading, among the others, to abnormally sustained supersaturation of concentrated bile enriched with cholesterol (due to hepatic cholesterol hypersecretion), and accelerated phase transitions of biliary cholesterol within a hypocontractile gallbladder (Portincasa et al., 2008a). Also, cholesterol gallstone disease is currently considered as the hepatobiliary expression of the metabolic syndrome, since it is often associated with obesity, type 2 diabetes, dyslipidemia, and hyperinsulinemia. During murine lithogenesis obtained in C57L mice susceptible to diet-induced cholesterol gallstones, a decrement of the gallbladder concentrating ability was associated with reduced expression of the gallbladder AQP1 and AQP8 water channels (van Erpecum et al., 2006). Alterations in mRNA levels of AQP1 and AQP4 were found in the gallbladder of Lepob mice undergoing leptin replacement (Swartz-Basile et al., 2007). Besides showing the characteristic obesity Lepob mice have enlarged gallbladder volumes and decreased gallbladder contractility, the latter indicating gallbladder stasis (Goldblatt et al., 2002). Studies are ongoing to check if similar phenomena also play a role in cholesterol gallstone disease in humans.

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7. Conclusions and future perspectives Studies on AQPs throughout the hepatobiliary epithelia are highly instructive, and provide important insights into complex molecular mechanisms by which biliary water is secreted and reabsorbed in concert with lipidic and non-lipidic compounds. Valuable acquisitions are being made regarding the pathophysiological involvement of AQPs in multiple diseases, namely cholestasis, liver cirrhosis, cholesterol cholelithiasis, polycystic liver and even microparasite invasion of intrahepatic bile ducts. Nevertheless, several aspects regarding the regulation and physiology of hepatobiliary AQPs remain to be fully assessed. While specific association of AQPs with liver cystic fibrosis awaits deeper analysis, the role of such water channels in hepatobiliary disorders with fluid imbalance such as sclerosing cholangitis should be addressed. Overall, this process should greatly enhance our understanding of bile formation and bile flow in health and disease. While investigating the ultimate pathophysiological role of AQPs in several clinical disorders (Calamita and Portincasa, 2007; Jeyaseelan et al., 2006; Goldblatt et al., 2002), novel pharmacological strategies might appear soon to treat bile secretory failures in which hepatobiliary epithelia are the target cell.

Acknowledgments This work was supported in part by Research Grants from Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) FIRB2003 and RBAU01RANB002 (P.P.), PRIN20089SRS2X_003 (G.C.), Italian National Research Council (CNR) Mobility Grant 2005 (P.P.), Regione Puglia (Rete di Laboratori Pubblici di Ricerca ‘‘WAFITECH’’) and Fondazione Cassa di Risparmio di Puglia (Ricerca Scientifica e Tecnologica) (G.C. and P.P.). We gratefully acknowledge the support from Centro di Eccellenza di Genomica in campo Biomedico ed Agrario (CEGBA) (G.C.), the European Society for Clinical Investigation (ESCI) and the Accademia Pugliese delle Scienze (P.P.). Due to space limitations, we apologize to those whose publications related to the discussed issues could not be cited.

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Wang, H.H., Portincasa, P., Wang, D.Q., 2008. Molecular pathophysiology and physical chemistry of cholesterol gallstones. Front. Biosci. 13, 401–423. Wang, D.Q., Cohen, D.E., Carey, M.C., 2009. Biliary lipids and cholesterol gallstone disease. J. Lip. Res. 50 (Suppl), S406–S411. Wang, H.H., Portincasa, P., Afdhal, N.H., Wang, D.Q., 2010. Lith genes and genetic analysis of cholesterol gallstone formation. Gastroenterol. Clin. North Am. 39, 185 (viii). Yakata, K., Tani, K., Fujiyoshi, Y., 2011. Water permeability and characterization of aquaporin-11. J. Struct. Biol. 174, 315–320. Yang, B., Song, Y., Zhao, D., Verkman, A.S., 2005. Phenotype analysis of aquaporin-8 null mice. Am. J. Physiol. Cell Physiol. 288, C1161–C1170. Yasui, M., Hazama, A., Kwon, T.H., Nielsen, S., Guggino, W.B., Agre, P., 1999. Rapid gating and anion permeability of an intracellular aquaporin. Nature 402, 184–187. Yokomori, H., Oda, M., Yoshimura, K., Kaneko, F., Hibi, T., 2011. Aquaporin-1 associated with hepatic arterial capillary proliferation on hepatic sinusoid in human cirrhotic liver. Liver Int. 31, 1554–1564. Yovchev, M.I., Grozdanov, P.N., Zhou, H., Racherla, H., Guha, C., Dabeva, M.D., 2008. Identification of adult hepatic progenitor cells capable of repopulating injured rat liver. Hepatology 47, 636–647. Zardoya, R., 2005. Phylogeny and evolution of the major intrinsic protein family. Biol. Cell 97, 397–414. Zollner, G., Trauner, M., 2008. Mechanisms of cholestasis. Clin. Liver Dis. 12, 1–26 (viii). Dr. Piero Portincasa is Professor of Internal Medicine at the Department of Biomedical Sciences and Human Oncology at the University Medical School of Bari, Italy. In 1985–1987, as Research Fellow supported by a Grant from the Italian Ministry of University, he studied with Prof. R.H. Dowling at Guy’s Hospital, London, UK. In 1993–1995, he was Research Fellow and Staff Member with Prof. G.P. vanBerge-Henegouwen and Dr. K.J. van Erpecum at the Academic Hospital of the University in Utrecht, The Netherlands, where he completed a Ph.D. Program. In 2005, he was supported by a Fellowship from the Italian National Centre for Research (CNR) for a stage with Dr. D.Q.-H. Wang and Prof. J.T. LaMont at Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, USA. He is an active member of several International Scientific Societies, and Journal Editorial Boards, Member of the Apulian Academy of Sciencies, President of the European Society for Clinical Investigation, Honorary Visiting Professor at the University of Cluj-Napoca, Romania, Councillor of the Apulian Section of Italian Society Internal Medicine, and Honorary Member of the Romanian Society Gastroenterology and Hepatology. He has been mentor and panelist in Ph.D. programmes shared with the University of Utrecht, The Netherlands, Coimbra and Porto, Portugal, and Saint Louis, USA. Dr. Portincasa’s major research interest is in the area of lipid metabolism and enterohepatic circulation with respect to mechanisms leading to cholelithiasis, fatty liver, and metabolic syndrome. He has been performing several translational studies focusing on transport of water and ions in the hepato-intestinal tract, and gastrointestinal motility. Giuseppe Calamita is Full Professor of Physiology at the Dept of Biosciences, Biotechnologies and Pharmacological Sciences at the University of Bari (Italy). After his Bachelor of Biological Sciences, in February 1986, he works as research trainee at the Institute of General Physiology at the University of Bari. In 1986, with a 14-months research fellowship by the European Community, he joins the laboratory of Jacques Bourguet at the Centre of Nuclear Studies (CEA) of Saclay (France) to work on the biophysical, biochemical and pharmacological characterization of the ADH-induced membrane water transport. His Ph.D. (1987–1990), 18 months of which done in France (CEA), is aimed at the identification of the ADH-induced water channels. In 1990, he becomes Assistant Professor (University of Bari) working on the isolation and biochemical characterization of renal water channels. He is PostDoc (1994–1996) at the laboratory of Peter Agre at the Dept Biological Chemistry of the Johns Hopkins University of Baltimore (USA), where he discovers the first aquaporin water channel, AqpZ, in Escherichia coli. Back to Bari, he starts his own research group. Presently, he is primarily focusing on the biophysics, physiology and pathophysiology of liver AQP8 and AQP9. Since 2008, he is also directing a network of Italian research laboratories aimed to construct biomimetic membranes incorporating AQPs for biotechnological applications.